[Histonet] Processing Schedule- ASP-6025

Fri May 10 11:46:25 CDT 2019

What are you using for your lab's own control. Was that tissue processed
recently? Do you have normal tonsil that you process  in-house regularly
that also stains poorly?

On Wed, May 8, 2019 at 9:16 AM Greg Dobbin via Histonet <
histonet at lists.utsouthwestern.edu> wrote:

> Great point John!
> We do attempt to have all of the specimen handling for our control tissue,
> match that of our specimens. However,
> tonsillectomies at our hospital are only performed on Thursdays. So we fix
> them for 24hrs (until Friday) then process overnight and remove
> from the processor on Saturday (we don't typically work Saturdays). And I
> have done this a couple of times but can I swear that this particular
> tonsil
> was handled that way? I cannot ...which I know, points to another
> deficiency! :-0  It could very well be that the fixation times of the two
> tonsils I am comparing are sufficiently different that they will require
> different retrieval times to produce stains of similar intensity.
>
> I also appreciate getting your opinion on the processing schedule. You have
> much more experience than I!! Thank you for your insights.
> All the best,
> Greg
>
> On Wed, May 8, 2019 at 10:50 AM John Garratt <john.garratt at ciqc.ca> wrote:
>
> > The processing schedule looks fine. I am thinking that if you are having
> > no problems with morphology on the H&E processing is not the issue. The
> > extended period in the warming draw is a good hypothesis.
> > I do have a question. How long was that particular piece of tonsil kept
> in
> > formalin before processing? Could over fixation be the issue?
> > I have found that maintaining tight control of control tissues is
> > important which includes minimum and maximum fixation time.
> >
> > John
> >
> > Sent from ProtonMail Mobile
> >
> >
> > On Tue, May 7, 2019 at 5:52 PM, Greg Dobbin via Histonet <
> > histonet at lists.utsouthwestern.edu> wrote:
> >
> > Fascinating Tony!
> > We don’t typically leave them in the warming drawer any longer than a
> > couple of hours, but maybe this particular tonsil was left linger for
> some
> > reason and no one thought anything of it?! Something to consider for
> sure!
> > Thanks.
> > Greg
> >
> > On Tue, May 7, 2019 at 9:37 PM Tony Henwood (SCHN) <
> > tony.henwood at health.nsw.gov.au> wrote:
> >
> > > Processing seems adequate.
> > >
> > > After processing, how long do they sit in the embedding centre block
> > > holding tank before embedding?
> > >
> > > We found that quite a few antigens were affected when we stored control
> > > tonsil in the embedding centre (dry) at 60oC for a few days before
> > > embedding. In summary:
> > >
> > > Antibody Clone Dried (Normal = 3+)
> > > CD4 4B12 0
> > > BOB-1 TG14 0
> > > CD3 LN10 1+
> > > CD79a JCB117 1+
> > > Oct-2 Oct-207 1+
> > > CD8 4B11 2+
> > > CD20 L26 3+
> > >
> > > So CD20 was unaffected but this process affected most of the antigens
> > with
> > > some losing antigen recognition by the antibody (eg CD4 and BOB-1).
> > >
> > > Another one of those pre-analytical issues we need to consider.
> > >
> > > And yes I am writing this up for submission!
> > >
> > >
> > > Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) |
> > > Principal Scientist; Adjunct Fellow, School of Medicine, University of
> > > Western Sydney; Visiting Lecturer, School of Life Sciences, Faculty of
> > > Science, University of Technology Sydney | Histopathology
> > > t: (02) 9845 3306 | f: (02) 9845 3318 | e:
> > tony.henwood at health.nsw.gov.au
> > > | w: www.schn.health.nsw.gov.au
> > > m:
> > >
> > >
> > > Cnr Hawkesbury Road and Hainsworth Street, Westmead, NSW Australia
> > > Locked Bag 4001, Westmead 2145, NSW Australia
> > >
> > > ♲ Please consider the environment before printing this email.
> > >
> > > -----Original Message-----
> > > From: Greg Dobbin via Histonet [mailto:
> histonet at lists.utsouthwestern.edu
> > ]
> > > Sent: Wednesday, 8 May 2019 5:07 AM
> > > To: histonet at lists.utsouthwestern.edu
> > > Subject: [Histonet] Processing Schedule- ASP-6025
> > >
> > > Hello colleagues,
> > > I recently stained (IHC) a section of normal tonsil from another
> facility
> > > with p16 and the resulting stain was better than the same stain on a
> > > section of my labs own normal tonsil control.
> > >
> > > This has led us to question our processing schedule. I am not concerned
> > > with our fixation because we fix everything for at least 24 hours in
> 10%
> > > formalin (commercially prepared) prior to processing.
> > >
> > > Does anything jump out at you as being a potential red flag in the
> > > following overnight protocol?
> > >
> > > - Formalin 15 mins; RT
> > > - Processing water 1 min; RT
> > > - ETOH 70% 30 mins; 35C
> > > - ETOH 80% 30 mins; 35C
> > > - ETOH 95% 30 mins; 35C
> > > - ETOH 100% 30 mins; 35C
> > > - ETOH 100% 40 mins; 35C
> > > - ETOH 100% 60 mins; 35C
> > > - Xylene 60 mins; 35C
> > > - Xylene 60 mins; 35C
> > > - Xylene 60 mins; 35C
> > > - Paraffin 60 mins; 57C; vacuum
> > > - Paraffin 60 mins; 57C; vacuum
> > > - Paraffin 60 mins; 57C; vacuum
> > >
> > > Our formalin is changed after every 1100 cassettes and the alcohol,
> > > xylenes and paraffins are managed similarly by the instrument. Our
> > specimen
> > > mix is a little of everything (skins, GIs, breasts, needle cores, gall
> > > bladders, gyne, etc).
> > >
> > > The one unknown (so far) in this story, is how the tonsil from the
> other
> > > laboratory was handled (ie the fixative used and for how long-I am
> > assuming
> > > 10% formalin).
> > >
> > > Obviously, many of you will have schedules that differ from this one,
> in
> > > any number of ways, but what I am looking for from you is your opinion:
> > *is
> > > there anything about this schedule that is particularly concerning?*
> > Thank
> > > you, Greg
> > >
> > >
> > > --
> > > *Greg Dobbin*
> > > 1205 Pleasant Grove Rd
> > > <
> https://maps.google.com/?q=1205+Pleasant+Grove+Rd&entry=gmail&source=g>
> > > RR#2 York,
> > > PE C0A 1P0
> > >
> > >
> > > *Everything in moderation...even moderation itself**!*
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> > >
> > --
> > *Greg Dobbin*
> > 1205 Pleasant Grove Rd
> > RR#2 York,
> > PE C0A 1P0
> >
> >
> > *Everything in moderation...even moderation itself**!*
> > _______________________________________________
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> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> >
> >
> >
> >
>
> --
> *Greg Dobbin*
> 1205 Pleasant Grove Rd
> RR#2 York,
> PE      C0A 1P0
>
>
> *Everything in moderation...even moderation itself**!*
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-- 

Tony Auge HTL (ASCP)CM QIHC
Cell: (651) 373-4768
Email: tony.auge at gmail.com